This project seeks to understand the potential of hemoglobin solutions (HbS), which are suitable for use as red blood cell substitutes, to cause liver toxicity. The toxicity of HbS may depend upon differences in preparations (e.g. cross-linking reagent, degree of polymerization) as well as the physiological and pathophysiological state of the patient. The liver may be especially susceptible to toxicity because it degrades hemoglobin metabolites such as iron, which causes oxidative injury and because it contains large numbers of resident macrophages (Kupffer cells). These cells (the reticuloendothelial system, RES) are activated to phagocytize foreign materials in the circulation. Although impure preparations of HbS undoubtedly activate the RES, it is not clear to what extend endotoxin-free, cross-linked preparations cause this activation. This is an important question because activation of RES stimulates Kupffer cells to secrete cytotoxic substances including superoxide anion and tumor necrosis factor-alpha (TNFalpha). Superoxide is a toxic reactive oxygen intermediate (ROI) which may also be produced directly by hemoglobin or its breakdown product, heme and iron. Therefore, the hypothesis to be investigated in this project is that ROI which are generated directly by oxidation of hemoglobin, heme, or iron synergize with ROI due to activation of Kupffer cells and together they may cause oxidative injury in hepatocytes. Coincident with these events, however, the gene expression of stress proteins such as heme oxygenase and nitric oxide synthase is up-regulated by these same potentially toxic intermediates. The toxicity which is manifested by HbS will depend upon the balance between activation of such protective proteins and the formation of ROI. Altered physiological states such as infection and ischemia may tip the balance toward toxicity because ROI generation is increased already by the pathophysiology. To investigate this hypothesis, we will infuse HbS into normal rats, volume depleted rats, rats pre-treated with endotoxin, and rats with ischemic levers. We will measure oxidative stress (as indicated by plasma GSSG levels), liver toxicity, the extent and nature of Kupffer cell activation or priming, the induction of heme oxygenase, nitric oxide synthase (potentially protective proteins) as well as cytokines such as TNFalpha, IL-1, and IL- 6. Activation of transcription factors known to be important in the oxidative stress response in the liver will also be determined. The contribution of Kupffer cells to these events will be evaluated by use of inhibitors of Kupffer cell activation. ROI formation in hepatocytes will be directly measured in primary cultures treated with HbS or Kupffer cell conditioned medium and HbS. Using these primary cultures, the effectiveness of anti-oxidants will be determined. This in vitro system will also be used to compare various preparations of HbS (e.g. chemically cross-linked HbS vs recombinant HbS.)